Antimicrobial-resistant bacteria are an emerging public health concern. This study aimed to determine the prevalence of antibiotic resistant strains of Escherichia coli cultured from wild herring gulls (Larus argentatus) and from human wastewater at Cape Cod, Massachusetts, USA. Bacterial sensitivity by disc diffusion was tested with seven antibiotics: ampicillin (10 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), gentamicin (10 µg), streptomycin (10 µg), sulfamethoxazole-trimethoprim (23.75/1.23 µg), and tetracycline (30 µg). Of the 49 wastewater isolates 59.2% percent were resistant to at least one antibiotic, and 40.8% demonstrated multi-drug resistance. Of the 115 gull isolates, 15.6% were resistant to one antibiotic, and 0.9% demonstrated multi-drug resistance. Ampicillin, streptomycin, and tetracycline resistant isolates were the most common type of resistance phenotypes in both gull and wastewater isolates. Strains of E. coli that exhibited resistance phenotypes were genetically analyzed to identify the presence of antibiotic resistance and virulence genes. Shared resistance genes (including blaTEM, strA, and tetB) were documented in both gull and wastewater E. coli samples. One or more virulence genes (including STa, iss, and tsh) were identified in 3/12 (25%) of the antibiotic resistant gull isolates. This research is one of the first studies documenting the genetic determinants of antibiotic resistance in wildlife of the United States.